Chapter 16

The Cytoskeleton

Eucaryotic cells contain protein fibers that are involved in- establishing cell shape- providing mechanical strength- cell movement- chromosome separation- intracellular transport of organelles

Protein fibers form the cytoskeleton and there are 3 typesof these protein filaments:- Actin filaments (also called microfilaments)- Intermediate filaments- Microtubules

In addition, a large number of accessory proteins, includingthe motor proteins, are required for the properties associatedwith each of these filaments

Each type of filament has distinct mechanical properties anddynamics, but certain fundamental principles are common toall.

The self-assembly and dynamic structure of cytoskeletal filaments

Some functions of actin filaments are:- to provide mechanical strength to the cell by forming a band under the plasma membrane- link transmembrane proteins to cytoplasmic proteins- form contractile ring during cytokinesis in animal cells- cytoplasmic streaming- generate locomotion in cells such as white blood cells and amoeba- Interact with myosin to provide force of muscular contraction

Microtubules participate in a wide variety of cell activities. Most involve motion that is provided by protein “motors” that use ATP. They determine the positions ofmembrane-enclosed organelles and direct intracellular transport. The migration ofchromosomes during mitosis and meiosis takes place on microtubules that make upthe spindle fibers.

Intermediate filaments provide mechanical strength and resistance to shear stress.There are several types of intermediate filaments, each constructed from one ormore proteins characteristic of it.

Keratins are found in epithelial cells, hair and nailsNuclear lamins form a meshwork that stabilizes the inner nuclear membraneNeurofilaments strengthen the long axons of neuronsVimentins provide mechanical strength to muscle and other cells

Nature 422, 741 - 745 (17 April 2003)

Intermediate filaments

Defective keratins lead to the epidermolysis bullosa simplex disorder

The tubulin and actin subunits assemble head-to-tail to create polar filaments

The structure of a microtubule and its subunit

The structure of an actin monomer and actin filament

Microtubules and actin filaments have two distinct ends that grow at different rates

Nucleation is the rate-limiting step in the formation of a cytoskeletal polymer

The time course of actin polymerization in a test tube

Filament treadmilling and dynamic instability are consequences of nucleotidehydrolysis by tubulin and actin

Treadmilling occurs at intermediate concentrations of free subunits

Dynamic instability

Actin filaments

Actin filaments nucleate most frequently at the plasma membrane and nucleation is regulated by external signals

Figure 16-34a Molecular Biology of the Cell (© Garland Science 2008)

Nucleation is catalyzed by a complex of proteins that include actin-related proteins (ARPs)

Differences on the sides and minus end prevent the ARPs from forming filaments on their own or with actin.

Figure 16-34b Molecular Biology of the Cell (© Garland Science 2008)

The ARP complex nucleates actin filament growth from the (-) end, allowing rapid elongation at the (+) end

The ARP complex can also attach to the side of another actinfilament while remaining bound to the (-) end of the filament

that it has nucleated

Figure 16-34c Molecular Biology of the Cell (© Garland Science 2008)

The ARP complex nucleates filaments more efficiently when it is bound to the side of a preexisting actin filament resulting in a filament branch that grows at a 70° angle

relative to the original filament

Figure 16-36 Molecular Biology of the Cell (© Garland Science 2008)

Actin elongation is mediated by formins

Formation of actin bundles (as opposed to the gel-like branched actin networks) is induced by formins, which are able to nucleate the growth of straight, unbranched filaments that can be cross-linked by

other proteins to form parallel bundles. Formins are dimeric proteins and each subunit has a binding site for an actin monomer.

Filament elongation is modified by proteins that bind to thefree subunits

Why does the soluble actin in cells not polymerized into filaments if the concentration of soluble actin is high (50-200 M)?

Although the Cc of actin monomers is 0.1 M, the actin is not polymerizedas it is bound to special proteins, such as thymosin. Actin monomers bound to thymosin are locked where they cannot associate with either the (+) endor (-) end of the actin filament.

How do cells recruit actin monomers from this sequestered pool and usethem for polymerization?

Recruitment depends on another monomer-binding protein profilin.

Profilin binds to the face of actin opposite the ATP-binding cleft.Actin-profilin can bind to the plus end of the actin filament but is unable tobind to the minus end.

Figure 16-37 Molecular Biology of the Cell (© Garland Science 2008)

Proteins that bind to the sides of actin filaments can eitherstabilize or destabilize them

Tropomyosin stabilizes actin filaments by binding simultaneouslyto seven adjacent actin subunits in one protofilament This preventsother proteins from binding to actin

Cofilin destabilizes actin filaments by forcing it to twist a little more tightly

Cross-linking proteins organize assemblies of actin filaments

Bundling and gel-forming proteins

Figure 16-29 Molecular Biology of the Cell (© Garland Science 2008)

Polymerization of tubulin nucleated by -tubulin ring complexes

Figure 16-30a Molecular Biology of the Cell (© Garland Science 2008)

Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008)

The centrosomeCell polarity including the organization of cell organelles, direction of membrane

trafficking, and orientation of microtubules is determined by microtubule-organizingcenters (MTOCs).

Microtubule-binding proteins (MAPs) organize microtubules and affect their stability. Some MAPs prevent or promote cytosolic microtubule polymerization;other MAPs organize microtubules into bundles or cross-link them to membranesand intermediate filaments or both.

Figure 16-40 Molecular Biology of the Cell (© Garland Science 2008)

tau-greenMAP2-orange

Figure 16-41 Molecular Biology of the Cell (© Garland Science 2008)

Figure 16-44 Molecular Biology of the Cell (© Garland Science 2008)

Actin-based motor proteins are members of the myosin superfamily

Myosin II

The myosin II bipolar thick filament

Direct evidence for the motor activity of the myosin head

Comparison of the domain structure of the heavy chains of some myosin types

Myosin VI is unique in moving towards the minus end of an actin filament

Kinesin and kinesin-related proteins

The structural similarity of myosin and kinesin indicates a common evolutionary origin

Dyneins are a family of minus-end directed microtubule motorsThey are composed of two or three heavy chains (that include the motor domain) anda variable number of light chains

Two major families of dyneins – cytoplasmic dyneins and axonemal dyneinsCytoplasmic dyneins found in all eucaryotic cells – important for vesicle trafficking and localization of the Golgi apparatus near the center of the cell Axonemal dyneins are highly specialized for rapid and efficient sliding movement of microtubules that drive the beating of cilia and flagella

Dynein requires the presence of a large number of accessory proteinsto associate with membrane-enclosed organelles

Homologs of the eucaryotic cytoskeleton in bacteria